Synthesis and biological evaluation of novel quinoline analogs of ketoprofen as multidrug resistance protein 2 (MRP2) inhibitors

Objective(s): A new series of quinoline analogs of ketoprofen was designed and synthesized as multidrug resistance protein 2 (MRP2) inhibitors using ketoprofen as the lead compounds. Materials and Methods: The cytotoxic activity of the compounds was evaluated againt two cancer cell lines including A2780/RCIS (MRP2-overexpressing ovarian carcinoma), A2780, drug-sensitive ovarian carcinoma using MTT assay. Compounds showing low toxicity in MTT test were selected to investigate their MRP inhibition activity. MRP2 inhibitory potency was evaluated by determination of the uptake amount of fluorescent 5-carboxy fluorescein diacetate (5-CFDA) substrate, by A2780/RCIS in the presence of the selected compounds. Mode of interaction between synthesized ligands and homology modeled MRP2 was investigated by MOE software. Results: Compound 6d, a 4-carboxy quinoline possessing dimethoxy phenyl in position 2 of quinoline ring, showed the most MRP2 inhibition activity among all the quinolines and more than the reference drug ketoprofen. MRP2 inhibition activity of compound 7d was less in comparison to that of compound 6d, indicating that carboxyl group in position 4 of quinoline may interact with MRP2. Docking studies showed that compound 7d methyl ester of 6d, interacted less compared to its parent 6d, which is consistent with biological results. Conclusion: This study indicates that 6- or 8-benzoyl-2-arylquinoline is a suitable scaffold to design MRP2 inhibitors. The position of benzoyl in quinoline ring is important in inhibition of MRP2. Generally, MRP2 inhibition activity of compound 7d was less in comparison to that of 6d, indicating that carboxyl group in position 4 of quinoline may interact with MRP2.


Introduction
Cancer is the reason of 25% of all deaths in developed countries (1). Although chemotherapy is the collective way for treatment of different cancers, it fails to treat most cancer patients with advanced disease due to the occurrence of drug resistance (2,3). One of the most essential mechanisms underlying MDR (multidrug resistance) is the overexpression of adenosine triphosphate (ATP)-binding cassette (ABC) super-family of transporters, which efflux both cytotoxic agents and targeted anticancer drugs using ATP driven energy (4). One important class of the ABC family is the human multidrug resistance-associated protein (MRP) family which comprises seven members. Numerous members of the MRP family especially MRP1 and MRP2 are complicated in the detoxification and defense of the host against xenobiotic materials. They are also expected to cause drug resistance by their ability in moving a wide range of anticancer drugs out of the cells and their occurrence in many different types of cancers (5).
Some authors have distinguished that NSAIDs can enhance antitumor activity of drugs, working as inhibitors of multidrug resistance proteins MRP or MDR1 (15,23). Enforced expression of COX-2 causes enhancement in MDR1 expression, so the use of COX-2 inhibitors to decrease action of MDR1 may enhance accumulation of chemotherapy agents and reduce resistance of tumors to anticancer drugs. It has already been described that NSAIDs inhibit MRP2 or MRP4 (24,25). Also, NSAIDs such as salicylate, piroxicam, ibuprofen, naproxen, sulindac, tolmetin, etodolac, dicrofenac, indomethacin, ketoprofen, phenylbutazone and celecoxib inhibit MRP1, MRP2 and/or MRP4 (25,26). A wide variety of NSAIDs like indomethacin and ketoprofen inhibited MRP2 and MRP4 facilitated methotrexate transport at concentrations to which the transporters may be unprotected under therapeutic conditions. As ketoprofen is a well-known MRP2 inhibitor (14) and some quinoline derivatives such as quinine (27)(28)(29)(30)(31) reported as MRP modulators, we designed novel 2-(aryl)quinolines possessing ketoprofen scaffold as MRP2 inhibitors. The rational for the design of these compounds is represented in Figure 2. The cytotoxic activity of the synthesized compounds was evaluated against two human cancer cell lines including A2780/ RCIS, cisplatin resistant human ovarian carcinoma (MRP2-overexpressing ovarian carcinoma); A2780, drug-sensitive ovarian carcinoma. Compounds showed low to moderate toxicity in MTT test were selected to investigate their MRP 2 inhibition activity. Moreover, trying to explain the results of biological experiments, docking studies of the selected compounds into the homology-modeled human MRP2, were carried out.

Chemistry
All reagents, chemicals and solvents used in this research were bought from Merck AG and Aldrich Chemical. Melting points were assessed using a Thomas-Hoover capillary apparatus. Infrared spectra were attained by a Perkin Elmer (Model 1420) spectrometer. To acquire 1 HNMR spectra Bruker FT-500 and 300 MHz instruments (Brucker Biosciences, USA) was used and A Bruker FT-300 MHz instrument was used to obtain 13 CNMR spectra. Chloroform-D and DMSO-D6 were used as solvents. Coupling constant (J) values are measured in hertz (Hz) and spin multiples are given as s (singlet), d (double), t (triplet), q (quartet), m (multiplet). The mass spectra were assessed using a 3200 QTRAP LCMS triple quadrupole mass spectrometer (LCMS) with an electrospray ionization (ESI) interface.

General procedure for preparation of 6-or 8-benzoyl-2-arylquinoline-4-carboxylic acid (Doebner reaction)
A solution of appropriate benzaldehyde (9.45 mmol) and pyruvic acid (1.25g, 14.3 mmol) in acetic acid (10 ml) was heated for 40 min then 2-or 4-aminobenzophenone (10 mmol) was added to the solution and refluxed overnight. After cooling, the formed precipitate was filtered and washed with hexane and recrystallized in ethanol.

General procedure for preparation of methyl 6-methoxy-2-arylquinoline-4-carboxylate
2-arylquinoline-4-carboxylic acid (4 or 6) (2 mmol) and potassium carbonate (10 mmol) were mixed. Methyl iodide (10 mmol) and acetone (10 ml) were added. The reaction mixture was refluxed. The progress of the reaction was checked (TLC). The reaction was finished after 5 hr. The solvent was evaporated in vacuo and water was added to the residual mixture. The product was collected by filtration and dried to obtain pure product.

MDR reversal studies
The MTT based assay was done by seeding 5000 cancer cells per 180 µl RPMI complete culture medium in each well of 96-well culture. Cisplatin was applied at concentrations of 12.5, 25, 50 and 100 μM in both A2780 and A2780/RCIS cancer cells in absence or presence of highest non-toxic concentrations of synthetized compounds. Cells were then incubated (37 ºC in 5% CO 2 incubator) for 48 hr. Then 25 µl of MTT solution (4 mg ml -1 ) were added to each well and then incubated at 37 ºC (3 hr). At the end of incubation, formazan crystals were dissolved in DMSO (100 µl) and plates were read in a plate reader (Synergy H4, USA) at 540 nm. This experiment was done in triplicate determination each time.

Flow cytometric efflux assay
Microplates containing1×10 6 resistant cells in each well were incubated with 10 μM of 5-CFDA for 60 min. After washing, synthetized compounds were added and the cells were further incubated (60 min). Cells were washed with ice-cold PBS (two times) and harvested. After centrifugation, supernatants were removed and cells suspended in ice-cold PBS. Samples were analyzed by a BD FACS Calibur Flow Cytometer (BD Biosciences, San Jose, USA). Fluorescence intensity of substrate accumulated in the cells was measured with FlowJo 7.6.1 data analysis software (Oregon, USA). Cells treated with ketoprofen were used as controls.

Molecular modeling
Mode of interaction between synthesized ligands and homology modeled ABCC2 (MRP2) was investigated by docking. 2D structure of chemicals was organized in Chem Draw Ultera 12.0 software and 3D structures were arranged by Chem Draw Ultra 12.0 software using molecular mechanic force filed pre-optimization monitored by MM2 calculation. Further modification such as polar hydrogen addition was achieved by MOE software. Synthesized chemicals were docked into the binding site of MRP2 by MOE software. The docking simulations were done using triangle matcher placement algorithm with London dG scoring function and force field as refinement method. For each compound, the topscore docking poses were selected for final ligand-target interaction analysis using LigX module in MOE Software.

Synthesis
A one-step Doebner reaction was used to make 2-arylquinoline-4-carboxylic acid derivatives. As shown in scheme 1, 2-or 4-aminobenzophenone 1, substituted benzaldehyde 2 and pyruvic acid 3 were refluxed in acetic acid to obtain 4-carboxy quinolines (4 and 6) (42) and then esterification of carboxylic acid group was performed using methyl iodide in acetone (43) to afford the novel quinoline-4methyl esters (5 and 7). The compounds were characterized by nuclear magnetic resonance, infrared spectroscopy and mass spectroscopy.

Biological evaluation In vitro cytotoxic effects
Mahdizadeh et al. (44) examined the basic level of the mRNA expression of MRP1 and MRP2 in A2780/ RCIS cells and sensitive parental A2780 cell line. They Scheme 1. Reagents and conditions: (a) acetic acid, reflux (b) K 2 CO 3 , CH 3 I, Acetone, reflux reported that the MRP1 mRNA level in the resistant cell line (A2780/RCIS) was 1.29 times more than its expression level in sensitive cells (A2780 cells). Also, their results displayed that the expression level of MRP2 mRNA in the A2780/RCIS (resistant cell line) was much more (13 times) than the MRP2 mRNA level in parental A2780 cells. To identify ideal MRP inhibitors reversing MDR at non-toxic concentrations, cytotoxicity of the quinoline compounds against parental sensitive A2780 cells and their resistant sublines A2780/RCIS cells which overexpress MRP2 was evaluated by MTT assay. Cisplatin and ketoprofen were selected as controls. Most of our compounds exhibited negligible or much lower cytotoxic effect in both cancer cells. As depicted in Table  1, four quinoline derivatives 5a, 6b, 6c and 7b showed moderate cytotoxic activity with IC 50 in the range of 31.95-84.41 μM. However, the other quinolines did not display cytotoxic activity at concentrations below 100 μM.

Reversal of MRP -mediated MDR by quinoline derivatives
The reversal of multidrug resistance by the new quinoline derivatives was evaluated in drug-resistant cancer cell line with overexpression of MRP2 (A2780/ RCIS). The multidrug resistant cancer cell lines are remarkably resistant to the corresponding substrate anticancer drugs. We determined the cytotoxicity of cisplatin, in A2780/RCIS, multidrug resistant ovarian carcinoma cells (MRP2-overexpressing ovarian carcinoma cell line) and A2780, drug-sensitive ovarian carcinoma cells. The resulting IC 50 values are shown in Table 2. Our compounds are two groups, the first group is 8-benzoyl quinoline derivatives and the second group which is the isomers of the first group is 6-benzoyl quinoline derivatives. Compounds 4c, 5a, 5b and 5c from the first group and 6d, 7a, 7b and 7d from the second group at 30 μM concentration (almost the highest common non-toxic concentration between all synthetized compounds) exerted MDR reversal, and increased the anticancer activity of cisplatin in the human MRP2 overexpressing cell line A2780/RCIS. Compound 7d from the second group possessing dimethoxy phenyl in position 2 of quinoline exerted the most MDR reversal activity, and enhanced the cytotoxicity of cisplatin more than the other quinolines.

Biological evaluation of the MRP2 inhibition
Compounds exerted MDR reversal, and enhanced the cytotoxicity of cisplatin in the human MRP2 overexpressing cell line A2780/RCIS, including 4c, 5a, 5b, 5c (from the first group), 6d, 7a, 7b and 7d (from the second group) were selected to investigate their MRP2 inhibition activity. MRP2 inhibition was evaluated by the determination of the uptake amount of the fluorescent 5-carboxy fluorescein diacetate (5-CFDA) substrate, by A2780/RCIS ovarian carcinoma cells overexpressing MRP2 in the presence of the selected compounds. Compounds from the first group 4c, 5a, 5b, 5c did not show significant MRP2 inhibitory activity at the concentration below 200 μM. Compound 4c showed the most potent MRP2 inhibitory activity in the first group in concentration of 500 μM in a dose-dependent manner (data not shown).
When compounds from the second group tested at the concentration of 30 μM, none of the compounds a Compound concentration required to inhibit tumor cell proliferation by 50%. Data are presented as the mean ± SD from the dose−response curves of three independent experiments except 6d and 7d were found to inhibit the efflux of 5-carboxyfloresin diacetate in A2780/RCIS cells (data not shown) compound 6d, a 4-carboxy quinoline possessing dimethoxy phenyl in position 2 of quinoline ring, showed the most potent MRP2 inhibition among all the tested quinolines in a dose-dependent manner and more than the reference drug ketoprofen. Surprisingly, compound 7d which exerted the most MDR reversal, and enhanced the cytotoxicity of cisplatin more than the other quinolines did not show the most MRP2 inhibition activity.

Docking studies
As X-ray crystallization of the MRP2 protein is not accessible, the lone structural information existing to date is a bacterial ABC transporter. A previous structure of a bacterial MDR-ABC transporter MsbA has been remoted due to incorrect topological assignments resulted from low resolution of the X-ray diffraction data (45). However, using the obsolete PDB entry a homology model of ABCC2/MRP2 has been built to predict the binding of our quinolines.

Homology modeling of ABCC2 (MRP2)
The 1545-amino acid human ABCC2 (MRP2) contains two nucleotide binding domains (46) and up to 17 transmembrane helices distributed within three transmembrane domains (TMD), 1, 2, and 3. It has been shown that the amino terminal TMD1 of ABCC1 is not essential for substrate transport. So experiments have focused on TMD2 and 3. Sequence for ABCC2 had swiss port entry Q92887. As it was described previously (47) (Figure 3).
To explain the results of biological experiments docking studies of ketoprofen, compounds 7d and 6d into the homology-modeled human MRP-2, were carried out ( Figure 3). As mentioned above, compound 6d, a 4-carboxy quinoline possessing dimethoxy phenyl in position 2 of quinoline ring, showed the most potent MRP2 inhibition among all the tested quinolines in a dose-dependent manner and more than the reference drug ketoprofen. Studying ligand interaction mode of 6d by LigX module of MOE software revealed that O and   interacted less compared to its parent 6d. As shown in Figure 4, the O atom of benzoyl group of 7d made hydrogen bond with ARG 393, the same as that of 6d, but esterification of 6d led to eliminate the hydrogen bonds with MET 595 and MET 598. Ketoprofen also interacted less than its derivatives 6d and 7d. As shown in Figure 4, the O atom of benzoyl group of ketoprofen made hydrogen bond with ARG 393, the same as that of 6d and 7d, O atom of hydroxyl group of ketoprofen, could form hydrogen bonds with MET 595, the same as 6d. Although ketoprofen possess carboxyl group which forms hydrogen bond with target, but its binding energy is more than its derivatives 6d and 7d (Table 3), indicating that the quinoline ring causes the carboxyl group to be placed in a direction that can interact more with the target, and also dimethoxy phenyl ring provided additional interactions with the target.

Discussion
This study indicates that 6-or 8-benzoyl-2arylquinoline is a suitable scaffold (template) to design MRP2 inhibitors. The position of benzoyl in quinoline ring is important in inhibition of MRP2. Generally, 8-benzoyl-2-arylquinolines showed more activity compared to their isomers (6-benzoyl-2-arylquinolines). Compound 6d, a 4-carboxy quinoline possessing dimethoxy phenyl in position 2 of quinoline ring, showed the most potent MRP2 inhibition among all the tested quinolines in a dose-dependent manner and more than the reference drug ketoprofen. MRP2 inhibition activity of compound 7d was less in comparison to that of 6d, indicating that carboxyl group in position 4 of quinoline may interact with MRP2. These hydrophobic interactions and hydrogen bonds formation of compounds with homology modeled MRP2 can describe inhibitory effect of these compounds. Docking studies showed that compound 7d methyl ester of 6d, interacted less compared to its parent 6d, which is consistent with biological results.

Conclusion
Benzoyl-2-arylquinoline is a suitable template to design MRP2 inhibitors. The position of benzoyl in quinoline ring is important in inhibition of MRP2. Carboxyl group in position 4 of quinoline may interact with MRP2. Docking studies described the biological results and is consistent with biological results.  Table 3. Results of molecular docking experiments for ketoprofen, compounds 7d and 6d